Resveratrol-induced autophagy is dependent on IP3Rs and on cytosolic Ca2+

Previous work revealed that intracellular Ca(2+) signals and the inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) are essential to increase autophagic flux in response to mTOR inhibition, induced by either nutrient starvation or rapamycin treatment. Here, we investigated whether autophagy induced by resveratrol, a polyphenolic phytochemical reported to trigger autophagy in a non-canonical way, also requires IP3Rs and Ca(2+) signaling. Resveratrol augmented autophagic flux in a time-dependent manner in HeLa cells. Importantly, autophagy induced by resveratrol (80μM, 2h) was completely abolished in the presence of 10μM BAPTA-AM, an intracellular Ca(2+)-chelating agent. To elucidate the IP3R's role in this process, we employed the recently established HEK 3KO cells lacking all three IP3R isoforms. In contrast to the HEK293 wt cells and to HEK 3KO cells re-expressing IP3R1, autophagic responses in HEK 3KO cells exposed to resveratrol were severely impaired. These altered autophagic responses could not be attributed to alterations in the mTOR/p70S6K pathway, since resveratrol-induced inhibition of S6 phosphorylation was not abrogated by chelating cytosolic Ca(2+) or by knocking out IP3Rs. Finally, we investigated whether resveratrol by itself induced Ca(2+) release. In permeabilized HeLa cells, resveratrol neither affected the sarco- and endoplasmic reticulum Ca(2+) ATPase (SERCA) activity nor the IP3-induced Ca(2+) release nor the basal Ca(2+) leak from the ER. Also, prolonged (4h) treatment with 100μM resveratrol did not affect subsequent IP3-induced Ca(2+) release. However, in intact HeLa cells, although resveratrol did not elicit cytosolic Ca(2+) signals by itself, it acutely decreased the ER Ca(2+)-store content irrespective of the presence or absence of IP3Rs, leading to a dampened agonist-induced Ca(2+) signaling. In conclusion, these results reveal that IP3Rs and cytosolic Ca(2+) signaling are fundamentally important for driving autophagic flux, not only in response to mTOR inhibition but also in response to non-canonical autophagy inducers like resveratrol.status: publishe